Hypotensive active peptides

ABSTRACT

The present invention provides a hypotensive composition comprising a peptide having the structure X-Y, Y-X, or a salt thereof, wherein X is proline or proline-proline and Y is arginine, lysine, arginine-arginine, lysine-lysine, arginine-lysine, or lysine-arginine, together with a pharmaceutically suitable diluent.

This is a divisional of co-pending application Ser. No. 635,219 filedJuly 27, 1984, now U.S. Pat. No. 4,585,757.

BACKGROUND OF THE INVENTION

The present invention relates to short peptides, two to four amino acidsin length, to be used as antihypertensive agents.

Hypertension is a serious health problem of epidemic proportions. It isestimated that 20% of the adult population of North America havesystemic arterial pressures above the accepted normal range. A fewdirect causes of hypertension, such as pheochromocytoma and primaryhyperaldosteronism, are amenable to direct therapeutic intervention.These instances are not the norm. In general, pharmacologic treatment ofhypertension involves treating the symptom. Therapy is directed only toa correction of the abnormal pressure. Yet, it is now clear that suchtherapy can favorably affect prognosis and thus greatly decrease therisk of death due to cardiovascular disease in the affected person.

Essential, or primary, hypertension is a poorly defined condition thatis diagnosed by exclusion. It is not known whether hypertension is adisease per se or simply the upper end of a continuous spectrum of bloodpressures in the population. In both normotensive and hypertensiveindividuals, vascular tone is controlled by sympathetic nerve fibers.Even in normotensive individuals, reduction of the vascular tone canreduce the blood pressure. Thus, abnormal sympathetic nerve function isprobably not responsible for the increased peripheral resistance thatcharacterizes primary hypertension.

Pharmacologic therapy of hypertension has generally been directed atpromoting salt excretion (diuretics) and decreasing vascular tone byboth direct (direct vasodilators) and indirect (adrenegic blockingagents) means. Diuretics are an effective means of reducing overallblood volume and thereby reduce blood pressure, yet their use,particularly in moderate to severe hypertension, is generally secondaryto other agents. Diuretics also tend to deplete serum potassium, whichcan have serious consequences in the cardio-compromised patient.

The indirect vasodilators are probably the most extensively used agentsfor mild to moderate hypertension. These agents, for the most part, acton some part of the sympathetic nervous system. Propranolol, a betaadrenergic blocker, is widely used in this country to varying degrees ofsuccess. Propranolol alone, though, provides poor control ofhypertension. Such therapy can also lead to various uncomfortable sideeffects including a prolonged reduction in cardiac output accompanied bydepression, impotence, and a pronounced lethargy. Other indirect actingagents, including Guanethidine, Pargyline, Methyldopa, Reserpine,Clonidine, and the ganglionic blockers, have also displayed varyingdegrees of success with numerous side effects and untoward reactions.

Similarly, problems with side effects are common with long term use ofdrugs that act directly on the vascular smooth muscle. Diazoxide is bothhyperuricemic and hyperglycemic. Nitroprusside is a powerful vasodilatorbut its effects are transient and the drug must be given by continuousinfusion. Hydralazine is used regularly even though the incidence ofuntoward reactions is very high.

In all of these agents, the main problems facing the physician areefficacy and side effects. In the area of antihypertensives, even minorside effects can result in the serious problem of patientnon-compliance. In terms of efficacy, the direct acting agents appear tooffer the best control of the hypertensive state yet their toxicitiesand other limitations tend to decrease their usefulness, particularly inmild to moderate cases. It is in the interest of medical science,therefore, to develop new approaches to the treatment of hypertension.

SUMMARY OF THE INVENTION

The present invention provides a hypotensive composition comprising apeptide having the structure X-Y, Y-X, or a salt thereof, wherein X isproline or proline-proline and Y is arginine, lysine, arginine-arginine,lysine-lysine, arginine-lysine, or lysine-arginine, together with apharmaceutically suitable diluent.

It also provides a method for alleviating hypertension in hypertensivemammals which comprises administering to the mammal an antihypertensiveeffective amount of a peptide having the structure X-Y, Y-X, or a saltthereof, wherein X is proline or proline-proline and Y is arginine,lysine, arginine-arginine, lysine-lysine, arginine-lysine, orlysine-arginine.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a series of 24 small peptides, two to four aminoacids in length, which represent a new class of therapeutically usefulantihypertensive agents. The general structure of the peptides includedin the present invention can be defined as XY or YX, where X is proline(Pro) or proline-proline; and Y is arginine (Arg), lysine (Lys),arginine-arginine, lysine-lysine, arginine-lysine, or lysine-arginine.This yields the following 24 possible combinations:

    ______________________________________                                        XY                 YX                                                         ______________________________________                                        Pro--Arg           Arg--Pro                                                   Pro--Lys           Arg--Pro--Pro                                              Pro--Arg--Arg      Lys--Pro                                                   Pro--Lys--Lys      Lys--Pro--Pro                                              Pro--Arg--Lys      Arg--Arg--Pro                                              Pro--Lys--Arg      Arg--Arg--Pro--Pro                                         Pro--Pro--Lys      Lys--Lys--Pro                                              Pro--Pro--Arg--Arg Lys--Lys--Pro--Pro                                         Pro--Pro--Lys--Lys Arg--Lys--Pro                                              Pro--Pro--Arg--Lys Lys--Arg--Pro                                              Pro--Pro--Lys--Arg Lys--Arg--Pro--Pro                                         Pro--Pro--Arg      Arg--Lys--Pro--Pro                                         ______________________________________                                    

The compositions of the present invention may be synthesized by knownpolypeptide synthesis methods. These peptides are then administered tothe hypertensive mammal in a suitable diluent, such as normal saline,the peptide in an amount effective to reduce the mammal's bloodpressure.

The present invention may be better understood by reference to thefollowing examples.

EXAMPLE 1

Corticotropin Inhibiting Peptide (CIP) contains the following amino acidsequence, the underlined sequences of which are the sequences which arethe subject of the present invention:

Human CIP (ACTH 7-38):Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu[M.W. 3659.68]

This CIP fragment was obtained from the Peninsula Laboratories, SanCarlos, CA. When administered to dogs, this peptide resulted indose-related hypotension.

The dog blood pressure assay was conducted according to the followingmethod:

Mongrel dogs of either sex weighing between 6-15 kg were anesthetized byintravenous injection of sodium pentobarbital (30 mg/kg). The femoralartery and vein were cannulated for the measurement of blood pressureand the injection of drugs, respectively. Arterial blood pressure wasdetermined with a Statham pressure transducer and was recorded on aGrass polygraph. Mean arterial pressure (MAP) was calculated for theperiods before and after each peptide injection. A tracheotomy wasperformed to insure a patent airway for spontaneous respiration.

Hypotensive screening studies involved injecting each dog with 0.1; 0.5;1.0; and 2.0 ug/kg. The CIP as supplied by Peninsula Laboratories was inthe lyophilyzed form. Prior to injection the CIP was dissolved insaline.

Table 1 summarizes the results obtained.

                  TABLE 1                                                         ______________________________________                                        Dose-dependent hypotensive activity of CIP                                    Dose (μg/kg)                                                                          Blood Pressure Decrease (mmHg)                                     ______________________________________                                        0.1         7                                                                 0.5        10                                                                 1.0        19                                                                 2.0        21                                                                 ______________________________________                                    

EXAMPLE 2

The above-described dog blood pressure assay was also used for adetermination of the antihypertensive effect of the following peptides,all of which were procured from Peninsula Laboratories. For eachpeptide, the amino acid sequences described by the present invention areunderlined:

    __________________________________________________________________________    Peptide                                                                              Amino acid sequence                                                    __________________________________________________________________________    Bradykinin                                                                            ##STR1##                                                              Xenopsin                                                                              ##STR2##                                                              Neurotensin                                                                           ##STR3##                                                              Substance P                                                                           ##STR4##                                                              __________________________________________________________________________

The maximum decrease in blood pressure of the hypotensive effects ofthese peptides in dogs by the assay described above in EXAMPLE 1 isshown in Table 2. Each datapoint represents the mean of 4 replications.

                  TABLE 2                                                         ______________________________________                                                     Dose       Maximum Decrease in                                   Hypotensive agent                                                                          (moles/kg) Blood Pressure (mmHg)                                 ______________________________________                                        Substance P  5 × 10.sup.-10                                                                     50                                                    Bradykinin   3 × 10.sup.-9                                                                      44                                                    Neurotensin  7 × 10.sup.-10                                                                     40                                                    Xenopsin     3 × 10.sup.-9                                                                      32                                                    ______________________________________                                    

EXAMPLE 3

The following peptides include amino acid sequences (underlined)described by the present invention:

    ______________________________________                                        Peptide   Amino acid sequence                                                 ______________________________________                                        Tuftsin                                                                                  ##STR5##                                                           Neurotensin (8-13)                                                                       ##STR6##                                                           CIP fragment                                                                             ##STR7##                                                           Contraceptive tetrapeptide                                                               ##STR8##                                                           ______________________________________                                    

When administered to a rat, each peptide exerted dose-relatedhypotensive effects. These results are shown in Table 3. Each datapointrepresents the mean of 4 replications.

The rat blood pressure assay was conducted as follows:

Sprague-Dawley rats of both sexes, weighing between 100 and 200 grams,were anesthetized with sodium pentobarbital and the right carotid arteryand jugular vein were cannulated with polyethylene tubing PE50. Thehypotensive peptide was administered and blood pressure was recorded inthe anesthetized rats as described above for dogs.

                  TABLE 3                                                         ______________________________________                                        Dose--Dependent Hypotensive                                                   Activity of Selected Peptides                                                        Blood Pressure Decrease (mmHg)                                                          Neurotensin                                                                              CIP    Contraceptive                              Dose (μg/kg)                                                                        Tuftsin (8-13)     fragment                                                                             tetra peptide                              ______________________________________                                        0.1               0                                                           1.0              13                                                           3.0              48                                                           10.0             63          1                                                50.0                        18                                                100.0                       35     2                                          200.0                              3                                          300.0                       38                                                500.0                              9                                          1000.0    1                        13                                         2000.0                             15                                         3000.0   12                                                                   5000.0   30                                                                   9000.0   46                                                                   ______________________________________                                    

EXAMPLE 4

The dipeptides proline-lysine and arginine-proline were also tested fortheir hypotensive action in rats. These dipeptides were synthesized byVega Biochemicals, Tucson, AZ, and tested in the rat blood pressureassay according to the same method described above in EXAMPLE 3. Theresults were as follows:

    ______________________________________                                                 Blood Pressure Change in mm Hg at dose of                            Dipeptide  1 mg/kg    2 mg/kg    3 mg/kg                                      ______________________________________                                        L Arg--L Pro.sup.A                                                                       -0.8 ± 0.8                                                                            -1.3 ± 0.8                                                                            -13.8 ± 4.4                               L Pro--L Lys.sup.B                                                                       +0.2 ± 2.6                                                                            -2.6 ± 1.9                                                                             -7.0 ± 3.4                               ______________________________________                                         .sup.A All values are average of four replications.                           .sup.B All values are average of five replications.                      

EXAMPLE 5

These dipeptides, L Arg-L Pro and L Pro-L Lys, were also tested in therat tail artery helical strip in vitro assay. This assay is conductedaccording to the following method:

Male and female Sprague-Dawley rats were anesthetized with pentobarbital(50 mg/kg) before removal of the tail artery. Upon isolation the arterywas placed in ice cold Krebs-Hensleit solution (KHS) which wasoxygenated with 95% O₂, 5% CO₂. The vessels were cut helically andstrips of approximately 1.5 cm were secured in a Sawyer-Bartlestonchamber containing KHS. The development of force of the helical stripswas measured with a Grass FT.03 force displacement transducer andrecorded on a Grass Model 79D polygraph. Isolated tail artery helicalstrips were equilibrated for one hour prior to addition of any peptide.In all cases, the strips were first contracted by adding argininevasopressin (AVP), to the bath. The peptide being studied was then addedto the bath and the degree of relaxation was determined.

The pure amino acids L-proline, L-arginine, and D-arginine were includedin this assay as controls.

The results were as follows:

    ______________________________________                                                  Tension change in gm at dose of                                     Dipeptide   100 mg/ml  200 mg/ml 500 mg/ml                                    ______________________________________                                        L Arg--L Pro                                                                              -0.07      -0.18     -0.34                                        L Pro--L Lys                                                                              -0.09      -0.16     -0.25                                        L Pro       0          0         0                                            L Arg       0          0         0                                            D Arg       0          0         0                                            ______________________________________                                    

EXAMPLE 6

Addition of lysine to the bradykinin molecule above results in thefollowing amino acid sequence: Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg.

When tested in the rat blood pressure assay this analog was even morehypotensive than bradykinin itself.

The above examples are provided by way of illustration only, and are notto be construed as a limitation on the scope of the invention defined bythe following claims.

What is claimed is:
 1. A hypotensive active peptide having the L-formamino acid sequence:

    ______________________________________                                        Pro--Lys;        Lys--Arg--Pro;                                               Pro--Arg--Arg;   Arg--Lys--Pro;                                               Pro--Lys--Lys;   Pro--Pro--Arg--Arg;                                          Pro--Arg--Lys;   Pro--Pro--Arg--Lys;                                          Pro--Lys--Arg;   Pro--Pro--Lys--Lys;                                          Pro--Pro--Arg;   Pro--Pro--Lys--Arg;                                          Pro--Pro--Lys;   Arg--Arg--Pro--Pro;                                          Lys--Pro--Pro;   Lys--Lys--Pro--Pro;                                          Lys--Lys--Pro;   Arg--Lys--Pro--Pro; or                                                        Lys--Arg--Pro--Pro,                                          ______________________________________                                    

and pharmaceutically acceptable salts thereof.
 2. A hypotensive activepeptide having the L-form amino acid sequence:Pro-Lys; Pro-Arg-Lys;Lys-Arg-Pro; or Lys-Arg-Pro-Pro,and pharmaceutically acceptable saltsthereof.
 3. A hypotensive active peptide having an L-form amino acidsequence:

    ______________________________________                                        Pro--Arg--Arg;   Pro--Pro--Arg--Arg;                                          Pro--Lys--Lys;   Pro--Pro--Arg--Lys;                                          Pro--Arg--Lys;   Pro--Pro--Lys--Lys;                                          Pro--Lys--Arg;   Pro--Pro--Lys--Arg;                                          Pro--Pro--Arg;   Arg--Arg--Pro--Pro;                                          Pro--Pro--Lys;   Lys--Lys--Pro--Pro;                                          Arg--Lys--Pro;   Arg--Lys--Pro--Pro; or                                                        Lys--Arg--Pro--Pro;                                          ______________________________________                                    

and pharmaceutically acceptable salts thereof.
 4. The hypotensive activepeptide of claim 3 which has the L-form amino acid sequence:Pro-Arg-Lysor Lys-Arg-Pro-Proand pharmaceutically acceptable salts thereof.